The focus of our research is the determinants of two transcriptional properties of steroid receptors: the dose-response curve of agonists and the partial agonist activity of antisteroids. The dose-response curve defines the EC50, or steroid concentration at which half-maximal response is seen, and is a crucial but poorly understood component of steroid hormone endocrinology. Differences in the EC50s of regulated genes provide a mechanism for differential expression by the common concentration of circulating steroid hormone in an organism. The partial agonist activity of antisteroids is an important consideration for limiting unwanted side effects during endocrine therapies by potentially allowing partial expression of genes other than the one targeted for suppression. While the EC50 and partial agonist activity were long considered to be invariant, it is now clear that they are not fixed and can be modulated by equilibrium interactions with a variety of factors. However, our initial findings suggest that the effects of specific factors are not the same with different receptors or even the same receptor with different genes. Thus, receptor- and gene-specific responses to cofactors may offer a mechanism whereby unequal responses can be elicited by different classes of receptors even though they can bind to the same DNA sequences of regulated genes.[unreadable] [unreadable] For classical steroid receptors, such as glucocorticoid receptors (GRs) and progesterone receptors (PRs), both agonist- and antagonist-bound GRs and PRs regulate gene transcription with the assistance of associated corepressors (nuclear receptor corepressor [NCoR] and silencing mediator of retinoid and thyroid hormone receptor [SMRT]) and coactivators (TIF2/GRIP1, SRC1, AIB1). These responses are considered to be mediated by receptor interaction domains (RIDs) in the middle and C-terminus of coactivators and corepressors respectively. Therefore, our recent finding that an amino terminal fragment of TIF2 (TIF2.0) competed for GR and PR interactions with a C-terminal RID-containing fragment of NCoR (NCoR-RID) in mammalian two hybrid assays was unexpected. We have used mammalian two-hybrid, pulldown, and coimmunoprecipitation assays to demonstrate that N-terminal GR sequences are sufficient to bind TIF2.0. In contrast, an N-terminal region of PR-B that is largely missing in the shorter PR-A is necessary but not sufficient for TIF2.0 binding. Mutagenesis studies establish that RID#1, but not RID#2, is necessary for NCoR binding to both GR and PR agonist and antagonist complexes. This suggests that the same element of NCoR-RID is involved in binding to GR and PR despite their differences in amino acid sequence. ChIP assays indicate that PR and NCoR each selectively localize to the enhancer element (PRE) of a transiently transfected PREtkluc reporter in the presence of both agonist and antagonist steroids while exogenous TIF2.0 causes a significant reduction in amount of NCoR associated with PRE in presence of the antiprogestin RU486. Importantly, exogenous TIF2.0 also inhibits the biological responses to added NCoR under the same conditions as the ChIP assays. We therefore propose that TIF2.0 inhibition of corepressor actions involves competitive binding to N-terminal regions of GRs and PRs. These results further suggest that both N-terminal and middle sequences of TIF2 contribute to its biological activity with GRs and PRs. Finally, we found that the full length TIF2, but not TIF2.0, binds to NCoR-RID. These results, in combination with our earlier demonstration that corepressor activity requires both N- and C-terminal domains of GR and PR, suggest that coactivators and corepressors have a more active role in modifying receptor transcriptional activities as opposed to simply binding to receptors in response to the steroid present in the LBD cavity.[unreadable] [unreadable] Another transcription cofactor that influences the biological responses of GRs is the novel protein STAMP that we described last year. STAMP modifies both the position of the dose-response curve (and the value of the EC50) for an agonist, and the amount of partial agonist activity for an antisteroid, for GR induced genes. STAMP also affects the ability of GR to repress gene expression. Thus, STAMP influences both aspects of GR-regulated gene expression: induction and repression. Our continued studies of STAMP reveal that the whole cell localization, as determined by Immunocytochemistry, changes with added glucocorticoid steroid from often cytoplasmic to almost exclusively nuclear. This is the expected result for a cofactor for GR-regulated gene expression. Both N- and C-terminal regions of GR appear to be required for binding to STAMP while the N-terminal half of STAMP is not required for GR binding or biological activity with GR. Interestingly, the biologically relevant C-terminal half of STAMP contains no known motifs, which suggests that the mechanism of STAMP action will also be unique. STAMP siRNAs reduce the protein levels, and reverse the biological activity with a transfected reporter gene, of transiently transfected STAMP cDNA. STAMP siRNAs also reduce the effects of endogenous STAMP and GR with 3 endogenous genes that are induced and one endogenous gene that is repressed. However, not all endogenous GR-induced genes are similarly affected by siRNA, which indicates that there is a yet unidentified element of specificity in the ability of STAMP to participate in the differential control of GR-regulated gene induction. [unreadable] [unreadable] As a result of the above studies, we have gained new molecular information about the modulation of the dose-response curve of agonists and the partial agonist activity of antisteroids by cofactors. These modulatory factors permit a continuum of responses and constitute new therapeutic targets for differential control of gene expression by steroid hormones during development, differentiation, homeostasis, and endocrine therapies. These combined findings contribute to our long-term goal of defining the action of steroid hormones at a molecular level and of understanding their role in human physiology.